Reduction of specific enterocytes from loss of intestinal LGR4 improves lipid metabolism in mice

Whether intestinal Leucine-rich repeat containing G-protein-coupled receptor 4 (LGR4) impacts nutrition absorption and energy homeostasis remains unknown. Here, we report that deficiency of Lgr4 (Lgr4iKO) in intestinal epithelium decreased the proportion of enterocytes selective for long-chain fatty acid absorption, leading to reduction in lipid absorption and subsequent improvement in lipid and glucose metabolism. Single-cell RNA sequencing demonstrates the heterogeneity of absorptive enterocytes, with a decrease in enterocytes selective for long-chain fatty acid-absorption and an increase in enterocytes selective for carbohydrate absorption in Lgr4iKO mice. Activation of Notch signaling and concurrent inhibition of Wnt signaling are observed in the transgenes. Associated with these alterations is the substantial reduction in lipid absorption. Decrement in lipid absorption renders Lgr4iKO mice resistant to high fat diet-induced obesity relevant to wild type littermates. Our study thus suggests that targeting intestinal LGR4 is a potential strategy for the intervention of obesity and liver steatosis.

Absorption of nutrients including fatty acid, amino acid and glucose by enterocytes substantially influences the global energy homeostasis 5 .Inhibition of lipid breakdown and absorption in intestine is efficient for the intervention of obesity and relevant metabolic disorders 6 .Traditionally, each absorptive enterocyte is considered being capable of absorbing multiple nutrients simultaneously.Our present study provides evidence challenging this classical concept, supporting the cellular heterogeneity of absorptive enterocytes.Using the single cell RNA sequencing analysis, we revealed that absorptive enterocytes contained three distinct populations: cells selective for long-chain fatty acid absorption, for carbohydrate absorption, and non-selective for long-chain fatty acid and carbohydrate absorption.Deficiency of Lgr4 specifically in intestinal epithelium (Lgr4 iKO ) significantly reduced the proportion of cells selective for long-chain fatty acid absorption.The reduction in enterocytes selective for long-chain fatty acid absorption substantially decreased lipid absorption, leading to subsequent improvement in global lipid and glucose metabolism.

Deficiency of intestinal Lgr4 reduces body weight and protects mice from HFD-induced obesity
Lgr4 mRNA was detected in a wide variety of tissues, with high abundance in hypothalamus and digestive organs such as pancreas, stomach, jejunum, ileum, and liver (Supplementary Fig. 1a).To determine whether intestinal LGR4 regulates energy metabolism, we generated Lgr4 iKO mice within which Lgr4 was specifically knocked out in intestinal epithelium (Supplementary Fig. 1b-d).Six-week-old male Lgr4 iKO mice and wild type (WT) littermates were fed normal chow diet (NCD) or 60% high fat diet (HFD) for 12 weeks.Compared with WT littermates, significant weight loss and smaller body size were observed in Lgr4 iKO mice fed either NCD or HFD (Fig. 1a, b).However, there was no significant difference in the body condition score (BCS) of Lgr4 iKO mice compared with WT littermates (Fig. 1c).Food intake was substantially reduced only in mice fed NCD but not in animals fed HFD, indicating that the weight loss is not entirely dependent on food intake (Supplementary Fig. 2a).Consistently, Lgr4 iKO mice displayed significantly less fat mass and lean mass (Fig. 1d, e).Fat weight and adipocyte size of subcutaneous white adipose tissue (sWAT) were significantly decreased.mRNA levels of beigeing marker gene Ucp1 was strikingly increased in sWAT of Lgr4 iKO mice fed HFD (Fig. 1f, g).Similar findings were observed in the epidydimal white adipose tissue (eWAT) (Fig. 1h, i).These results suggest that deficiency of intestinal Lgr4 reduces body weight and protects mice from HFD-induced obesity.
To further elicit the reason of weight loss in Lgr4 iKO mice, cold exposure and metabolic cage experiments were performed.As shown in Supplementary Fig. 2b, c, rectal body temperature during the 4 °C cold exposure, physical activity, and respiratory quotient (RQ = VCO 2 / VO 2 ) were not significantly altered by deficiency of intestinal Lgr4.These results indicate that weight loss of Lgr4 iKO mice was unlikely due to the alteration in thermogenesis and energy expenditure.

Deficiency of intestinal Lgr4 protects mice from HFD-induced liver steatosis
Next, we examined hepatic lipid metabolism in Lgr4 iKO mice.Liver weight and plasma triglyceride level were significantly decreased in Lgr4 iKO mice fed NCD (Fig. 2a-c).In Lgr4 iKO mice fed HFD, liver weight, plasma and hepatic triglyceride contents, and steatosis evidenced by H&E and oil red O staining were all decreased (Fig. 2d-f).These results suggest that deficiency of intestinal Lgr4 protects mice from HFDinduced hepatic steatosis.Interestingly, hepatic genes relevant to lipogenesis, lipid transport, and β-oxidation remained largely unchanged (Supplementary Fig. 3).The only exception was the upregulation of Pparα (Supplementary Fig. 3).These results indicate that deficiency of intestinal Lgr4 decreases hepatic lipid deposition likely via an extrahepatic mechanism.

Deficiency of intestinal Lgr4 decreases lipid absorption
The primary function of intestine is nutrient absorption.To assess the alteration of lipid absorption, we collected feces from mice, extracted total lipid, then measured fecal triglyceride levels.As shown in Fig. 3a, fecal triglyceride levels were significantly increased in Lgr4 iKO mice fed either NCD or HFD.Since HFD-feeding may alter lipid absorption, mice fed NCD were used in the following experiments.Plasma triglyceride levels and AUC of oral lipid tolerance test (OLTT) were substantially reduced in Lgr4 iKO mice after olive oil gavage (Fig. 3b).Oil red O staining showed that lipid droplets were remarkably decreased in the jejunum of Lgr4 iKO mice 2 h after gavage with 200 μL of olive oil (Fig. 3c).Examination of key intestinal lipid transporters revealed that both mRNA and protein levels of FATP4 and CD36 were substantially reduced (Fig. 3d-f).These results indicate that deficiency of intestinal Lgr4 decreases the expression of lipid transporters, leading to subsequent reduction in lipid absorption.
We next used MODE-K cells as an in vitro model to examine the effects of LGR4 on lipid absorption.Seventy-two hours after Lgr4 siRNA treatment, mRNA and protein levels of LGR4 and FATP4 in MODE-K cells were substantially reduced (Fig. 3g, h).These alterations were associated with a reduction in the concentration of intracellular triglyceride in MODE-K cells treated with a mixture of oleic acid and palmitic acid (Fig. 3i, P = 0.0548).Consistently, intensity of BODIPY fluorescence was also reduced (Fig. 3j).Next, we verified the effect of Lgr4 deletion on lipid uptake in intestinal organoids.The growth process of intestinal organoids was showed in Supplementary Fig. 4a.We found a significant decrease of Fabp1 and Fatp4 mRNA level (Supplementary Fig. 4b) and uptake of BODIPY (Supplementary Fig. 4c) in Lgr4-deficiency intestinal organoids.These results suggest that Lgr4 knockdown decreases lipid uptake.

Intestine-specific knockdown of Lgr4 improves glucose tolerance
In addition to absorption of lipid, we examined whether deficiency of Lgr4 would affect intestinal absorption of carbohydrate.After intraperitoneal injection of glucose, Lgr4 iKO mice exhibited improved glucose tolerance, particularly in the HFD group (Fig. 4a, b).Further, insulin resistance index (HOMA-IR) was decreased and insulin sensitivity index (HOMA-IS) increased in Lgr4 iKO mice fed HFD (Fig. 4c), suggesting that the increment in glucose tolerance may be attributed to improved peripheral insulin sensitivity.Interestingly, levels of blood glucose after oral glucose administration were not significantly altered compared with littermate control mice (Fig. 4d, e).The differential results of OGTT and IPGTT indicates that intestinal glucose absorption was increased in Lgr4 iKO mice.As shown in Fig. 4f, g, both mRNA and protein levels of GLUT2 were substantially elevated.These results suggest that deficiency of intestinal Lgr4 increases glucose absorption via up-regulation of glucose transporter, GLUT2.

Deficiency of intestinal Lgr4 decreases enterocytes selective for long-chain fatty acid absorption while increasing carbohydrateabsorptive enterocytes
Intestinal epithelial populations are crucial for dietary lipid and carbohydrate absorption.To determine the mechanism underlying the decrease of lipid absorption and concurrent increase of carbohydrate absorption, we analyzed the cellular heterogeneity of intestinal epithelia using the single cell RNAseq.After quality control of data filtering, intestinal epithelial populations were re-clustered using Seurat.According to the reported marker genes 7 , stem cells, TA cells, absorptive enterocytes, goblet cells, Paneth cells, enteroendocrine cells and tuft cells were defined (Fig. 5a, b).To further elucidate the order of differentiation between the epithelial populations, pseudotime analysis was analyzed and the cell markers of each population were projected on pseudotime axis.As shown in Fig. 5c, the differentiation of each population is well in line with the known order.These results confirm the accuracy for our definition of intestinal epithelial populations.
To investigate the cellular heterogeneity, absorptive enterocytes were re-clustered.Long-chain fatty acid-absorptive, and carbohydrateabsorptive enterocytes were defined by the expression of Cd36 and Fatp4、Glut2 and Sglt1 respectively (Fig. 5d).The statistical analysis for the proportions of absorptive cells revealed a decrease in enterocytes selective for long-chain fatty acid absorption and an increase in enterocytes elective for carbohydrate absorption in intestine of Lgr4 iKO mice (Fig. 5e).stem cells and TA cells 8 .To investigate the effect of intestinal LGR4 on stem cell differentiation, the proportions of each intestinal epithelial population was analyzed via single cell RNAseq.According to analysis of the number, the marker genes expression and specific staining, we found that deficiency of intestinal Lgr4 increased the proportion of absorptive enterocytes and TA cells, while decreasing the proportion of stem cells, goblet cells, Paneth cells, enteroendocrine cells and tuft cells (Supplementary Figs. 5, 6 and Fig. 6a).This observation was further confirmed by experiments using organoids derived from WT and Lgr4 deficient mice (Supplementary Fig. 6l).Consistent with the decrement in the number of Paneth cells and Tuft cells, intestinal barrier disruption (Supplementary Fig. 7a-d) and alteration of the microbiota (Supplementary Fig. 8) were observed in Lgr4 iKO mice.In addition, Lgr4 deficiency supressing the apoptosis indicates that the survival time of enterocytes is increased (Supplementary Fig. 7e).
Since the differentiation of intestinal stem cells into absorptive and secretory progenitors is regulated by Notch and Wnt signaling respectively 9,10 , we next examined the alteration of these two signaling pathways in Lgr4 iKO mice.As shown in Fig. 6b and c, cells highly expressing the absorptive progenitor genes (Ccnb1, Cdc20, Cenpa, Cdkn3, Ube2c, Aurka and Ccna2) substantially increased, whereas cells highly expressing the secretory progenitor gene (Dll1) decreased.Consistently, mRNA levels of absorptive progenitor marker and secretory progenitor marker genes were strikingly increased and decreased respectively (Fig. 6d) in intestine of Lgr4 iKO mice.These results indicate that deficiency of intestinal Lgr4 increases proportion of absorptive progenitors while concurrently decreases proportion of secretory progenitors.
The differentiation of secretory progenitors is activated by Wnt-βcatenin signaling, whereas the differentiation of absorptive progenitors depends on Notch signaling 9,[11][12][13][14] .Consistently, cells highly expressing the Wnt target genes such as Sox9, Sox4, Ascl2, Myc, Ube2c, and Lgr5, and the UMI value of these genes were decreased in Lgr4 iKO mice (Fig. 6e).Further, nuclear translocalization of β-catenin was decreased in intestine epithelia of Lgr4 iKO mice (Fig. 6f).mRNA levels of Math1, the downstream target of β-catenin was decreased (Fig. 6g).On the other hand, cells highly expressing Notch related genes such as Notch1, Notch4, Dtx1, Dtx3, Dll4, and Mfng and the UMI value of these genes were significantly increased (Fig. 6h).mRNA and protein levels of HES1, the downstream target of Notch was increased (Fig. 6i).As shown in Fig. 6j, activation of Notch signaling by VPA significantly suppresses the Wnt signaling evidenced by the decrement of its relevant target genes.This observation suggests a link between Wnt and Notch signaling.In addition, inhibition of Wnt signaling substantially decreases expression of lipid uptake genes Fatp4 in MODE-K cells (Fig. 6k).This observation suggests that inhibition of Wnt signaling may decrease lipid absorption.These results indicate that LGR4 regulates the differential differentiation of intestinal stem cells into absorptive and secretory cells via Notch and Wnt signaling pathways.Next, we further explored the relationship between LGR4 and Notch signaling in our research.In MODE-K cells, we quantified the mRNA levels of genes in Notch signaling, and found that levels of Psen1, Aph1a and Nedd4 were significantly changed (Supplementary Fig. 9a).We then further confirmed the increment of PSEN1 mRNA (Supplementary Fig. 9b) and protein levels (Supplementary Fig. 9c) in IEC6 cells.PSEN1 is an active component of γ-secretase, which is responsible for the cleavage of Notch and next-step function of NICD.These observations suggest that deficiency of LGR4 enhances the expression of Psen1 and thus stimulating the function of Notch signaling.The canonical pathway of LGR4 function is mediated by the nuclear translocation of β-catenin and then the transcriptional regulation of TCF/LEF transcription factor family.Using UCSC Genome Browser Home and JASPAR database, we predicted that TCF7L2 may bind to the promoter region of Psen1.Therefore, we constructed pcDNA3.4-Tcf7l2and pGL3-Psen1 promoter plasmids and cotransfected them with pRL-TK into 293T cells.We found that Psen1 relative luciferase activity was significantly reduced in condition of Tcf7l2 overexpression (Supplementary Fig. 9d).In organoids, Lgr4 deficiency increased Psen1 mRNA level (Supplementary Fig. 9e).Suppression of Wnt signaling by IWR-1 and activation of Notch using VPA mimicked, to some extent, the effects of Lgr4 deficiency on differentiation of intestinal stem cells to lipid absorptive cells (Supplementary Fig. 9f and Supplementary Fig. 9g).Further, suppression of Notch signaling using DAPT attenuated the differentiation of intestinal stem cells into lipid absorptive cells (Supplementary Fig. 9h-j).Together, these results suggest that deficiency of LGR4 may activate Notch signaling via βcatenin and TCF7L2 mediated transcriptional regulation of Psen1.

Discussion
LGR4 is abundantly expressed in digestive organs 15,16 .Previous studies have been focused on its role in intestinal development [17][18][19] .Our studies extend the physiological functions of intestinal LGR4 to modulation of lipid absorption and lipid homeostasis.Using the genetic approach to knockdown the Lgr4 gene specifically in intestinal epithelia, we demonstrate the metabolic benefit induced by deficiency of intestinal Lgr4.Knockdown of Lgr4 in intestinal epithelia rendered the mice resistant to HFD-induced obesity and its related metabolic disorders.Significant reduction in adiposity and liver steatosis was observed in Lgr4 iKO mice.The improvement in lipid metabolism appears not to be attributed to energy expenditure because coldexposure induced thermogenesis, physical activity and respiratory quotient were not altered.Rather, it is the reduction in lipid absorption that protects Lgr4 iKO mice from HFD-induced lipid disorders.Shortly after administration of lipid, plasma levels of triglyceride and lipid droplets inside the intestinal epithelia were substantially lower in Lgr4 iKO mice relevant to the wild type littermates.On the other hand, fecal levels of lipid were increased in the Lgr4 iKO transgenes.All these observations suggest the significant reduction of lipid absorption in Lgr4 iKO mice.The metabolic benefit of the decreased lipid absorption is significant.Mice with intestinal epithelial knockout of Lgr4 were The observation that deficiency of intestinal Lgr4 reduces intestinal lipid absorption while increasing glucose absorption suggests the functional significance of intestinal LGR4 in determining the selective absorption of nutrients.Each individual intestinal epithelial cell is classically considered to be capable of absorbing multiple nutrients such as glucose, fatty acid and amino acid without selection.Our study provides evidence challenging this concept.Using Cd36 and Fatp4, and Glut2 and Sglt1 to define enterocytes selective for absorption of longchain fatty acid or carbohydrate, we have revealed the heterogeneity of intestinal absorptive cells.Three distinct populations of absorptive cells are present in intestinal epithelia, including cells selective for absorption of long-chain fatty acid, carbohydrate, or both.Interestingly, the decrement in enterocytes selective for absorption of longchain fatty acid was associated with the concurrent increment in enterocytes selective for absorption of carbohydrate.The physiological significance of this concurrent alteration is unknown but may be related to protection against the uncontrolled insufficiency of energy absorption.Our study thus provides evidence supporting the cellular heterogeneity of absorptive enterocytes.Consistently, a recent spatial transcriptomics study has found that the expression of genes related to carbohydrate and lipid transport in absorptive cells differs in spatial distribution location 20 .This observation suggests the spatial heterogeneity in absorptive enterocytes absorbing various nutrients.
Global knockout of Lgr4 has been reported to block the terminal differentiation of Paneth cells only 4 , while there is no evidence for its effect on the differentiation of absorptive, enteroendocrine, and goblet-cell lineages.Surprisingly, our studies reveal that intestinespecific deletion of Lgr4 reduced proportion of enteroendocrine, tuft and goblet cells in addition to Paneth cells.These results indicate that LGR4 is crucial for the differentiation of secretory enterocytes.LGR4 binds directly to endogenous ligands such as RSPO1/2/3/4, Norrin, Nidogen-2, and RANKL [21][22][23][24][25] .Downstream signaling mediated by LGR4 primarily includes cAMP/PKA, Wnt/β-catenin, Gαq/GSK3β and Gαq/ PKCα signaling pathways.In addition, LGR4 can regulate AKT, extracellular signal-regulated kinase (ERK), and nuclear factor kappa-B (NF-κB) pathways 26 .However, how these signaling pathways regulate intestine epithelium remains unclear, and needs further exploration.Previous studies have demonstrated that differentiation of secretory occurs via activation of Wnt 9,10 .Consistently, deficiency of intestinal Lgr4 inhibits Wnt signaling in our study.On the other hand, deficiency of intestinal Lgr4 activates Notch signaling, leading to subsequent increment in the proportion of absorptive enterocytes.This observation is in line with previous report demonstrating that activation of Notch signaling is required for the differentiation of absorptive enterocytes [11][12][13][14] .As a limit of this study, the molecular mechanism by which LGR4 determines the selective differentiation of absorptive enterocytes is unknown.Our data suggest that deficiency of LGR4 may activate Notch signaling via β-catenin and TCF7L2 mediated transcriptional regulation of Psen1.Further experiment should explore whether the interaction between Notch and Wnt signaling pathways requires Psen1 using the genetic approach.
In summary, our study demonstrates that ablation of Lgr4 gene in intestinal epithelium affects stem cell differentiation via the activation of Notch and concurrent suppression of Wnt signaling pathway.This bias of stem cells differentiation results in reduced proportion of lipidabsorptive enterocytes, leading to decrement of lipid absorption and subsequent improvement in glucose and lipid metabolism (Fig. 7).Our results reveal the cellular heterogeneity of intestinal absorptive cells and the crucial role of intestinal LGR4 in controlling the differentiation of enterocytes selective for absorption of lipid.Our study thus suggests that targeting intestinal LGR4 may provide a potential strategy for the intervention of obesity and liver steatosis.

Animals and treatment
All experiments were conducted in strict accordance with the Guide for the Care and Use of Laboratory Animals prepared by the National Academy of Sciences (NIH publication 86 -23, revised 1985).Experimental protocols were approved by the Animal Care and Use Committee of Peking University.Villin-Cre mice were bred with Lgr4 flox/flox mice (from Helmholtz Zentrum, Germany) to generate Lgr4 iKO mice, within which Lgr4 was specifically knocked out in intestinal epithelium.Six-week-old male Lgr4 iKO mice and wild type (WT) littermates were fed normal chow diet (10 kcal% fat；D12450H；Research Diets) or high fat diet (60 kcal% fat；D12492；Research Diets) for 12 weeks.Animals were housed in a standard environment (22 ± 2 °C, humidity at 50 ± 15%) with 12 h light and 12 h dark cycle.Food and water were freely Results were expressed as mean ± SD. g mRNA levels of downstream genes of the Notch signaling pathway (Math1 and Hes1) detected by RT-qPCR.n = 5 for WT, and 6 for Lgr4 iKO .Results were expressed as mean ± SD. h Pseudotime showing Notch related genes expression (left) and heatmap showing UMI value (right).i Western blot and quantification of HES1 protein levels.β-actin was used as loading control.Results were expressed as mean ± SD. j Wnt and Notch related genes of the mouse small intestinal epithelial cell line MODE-K cells treated with the Notch activator VPA (P4543-25G, Sigma).*P < 0.05 vs NC. n = 3. Statistical analysis by two-way ANOVA with Šídák's multiple comparisons test.* P = 0.0001 for Axin2, * P = 0.0035 for Ccnd1, * P = 0.0003 for Hes1, * P < 0.0001 for Hey1.k mRNA levels of lipid absorption markers of MODE-K cells treated with the Wnt inhibitor IWR-1 (HY-12238, MedChemExpress).*P < 0.05 vs NC. n = 3. Statistical analysis by two-way ANOVA with Šídák's multiple comparisons test.* P = 0.0178.accessible except for the fasting experiment.At the end of the experiment, following tissue samples were harvested: plasma, intestine, liver, epididymal white adipose tissue (eWAT), and subcutaneous white adipose tissue (sWAT).

Tissue sample preparation and histological analysis
The dissected tissues were fixed with 4% paraformaldehyde in PBS for 24 h at 4 °C and stored in 20% sucrose phosphate buffer.Samples were embedded in paraffin or OTC and sectioned at a 3μm thickness (or 10μm for oil red O staining).H&E, immunohistochemical or oil red O staining were performed following general protocols.For the detection of intestinal Alkaline Phosphatase (ALPI), staining was performed according to the manufacturer's instructions (G1480, Solarbio, China).Periodic Acid Schiff (PAS) staining was performed according to the manufacturer's instructions (C0142, Beyotime, China).For Grimelius technique to stain enteroendocrine cells, deparaffinized and rehydrated samples were incubated in a preheated silver solution at 60 °C for 3 h.Afterwards, sections were washed in distilled water.then incubated in the 45 °C preheated reducing solution (1 g hydroquinone +2.5 g anhydrous sodium sulfite +100 ml ultra-pure water) for 1 h.Afterward, sections were washed in distilled water, dehydrated, transparentized and inspected by light microscope.

Oral glucose tolerance tests (OGTT) and Intraperitoneal glucose tolerance test (IPGTT)
Mice fasted for 16 h were orally gavaged (for OGTT) or intraperitoneally injected (for IPGTT) with glucose at a dose of 3 g/kg body weight.Blood was collected from the incision at the tip of the tail at 0, 15, 30, 60, 90, and 120 min after glucose administration, and the glucose concentration was immediately measured by a glucometer (Acuu-Chek Active, Roche, Germany).

Oral lipid tolerance test (OLTT)
Mice were fasted for 16 h before oral administration of olive oil (200 μL).Blood was collected from inner canthus at 0, 1, 2, 4, and 8 h after olive oil administration, and serum triglyceride were measured by colorimetry.

Western blot and quantitative RT-PCR
Protein was quantitated and loaded onto an SDS-PAGE gel and transferred to a PVDF membrane.Membrane was blocked with 5% nonfat dry milk in TBST at room temperature for 1 h, then incubated with the primary antibody overnight at 4 °C.IRDye-labeled secondary antibodies were used to detect specific reactions and visualized with the Odyssey infrared imaging system.The relative level of protein was assessed using Image J software.
RNA was isolated from tissues using Trizol, followed by reverse transcription.Quantitative real time-PCR was performed using SYBR green in Agilent AriaMx real-time PCR system.Supplementary Table 1 showed the primer sequences involved in this study.mRNA levels were normalized to the geometric mean value of reference genes (Hprt, Rpl32 and Tbp) or β-actin.
Cell culture and siRNA transfection MODE-K cells were cultured in a humid atmosphere (5% CO 2 ) using RPMI-1640 culture medium supplemented with 10% FBS and 1% penicillin/streptomycin at 37 °C.IEC-6 cells and 293T cells were cultured in a humid atmosphere (5% CO 2 ) using DMEM culture medium supplemented with 10% FBS and 1% penicillin/streptomycin at 37 °C.Cells were seeded in 12-well plate and grown until 70% confluency before transfection.Cells were washed with sterile phosphate-buffered saline (PBS) before transfection, then incubated with 50 nM Lgr4 siRNA or non-targeting Control siRNA (Synbio Technologies, Suzhou, China) for 48 h in a volume of 1 ml/well.Cells were then treated with mixture of oleic acid (0.6 mmol/l) and palmitic acid (0.2 mmol/l) or BODIPY-C 12 long-chain fatty acid (Invitrogen, Carlsbad, CA, USA) before harvesting for lipid absorption analysis.

Isolation of intestinal organoids
Mice were sacrificed, and then a proper length of intestine was taken out and washed for several times.Split the intestinal cavity and scrape the villus with a blade.Cut the intestine into pieces and incubate them in PBS containing 5 mM EDTA for 30 min.Vortex and filter through a cell sieve with a diameter of 70 μm to obtain crypts.Centrifuge and resuspend the crypts with the mixture of IntestiCult™ Organoid Growth Medium (06000, STEMCELL Technologies, Canada) and Matrigel (356234, Corning, USA).Seed the mixture in 24-well plate and put it in the cell incubator for 20 min to allow Matrigel to solidify, and then add appropriate amount of OGM into the plate and culture in the cell incubator.

Dual luciferase reporter gene assay
Mouse Tcf7l2 CDS region was cloned to pcDNA3.4 vector and 1440bp-1930bp upstream mouse Psen1 gene region (predicted binding site, Chromosome 12, NC_000078.7 (83732996.83733555)) was cloned to pGL3-basic vector.Cotransfected the plasmids together with pRL-TK (E2241, Promega, USA) into 293T cells.Harvest the cells after 24 h and conducted dual luciferase reporter gene assay using a kit (KGAF040, KeyGEN BioTECH, China) following the manufacturer's instructions.Preparation of single-cell suspension of intestinal epithelium The intestine was dissected longitudinally, minced, washed with PBS, then digested with 10 mmol/l EDTA on ice for 30 min.Cell suspension was filtered through 70 μm nylon mesh, centrifuged at 800 rpm for 5 min, then incubated with pre-warmed single cell digest containing dispase (1.67 U/ml) and DNaseI (0.01 mg/ml) at 37 °C.Cell suspension was then washed with PBS and filtered through 70 μm nylon mesh to remove the cell aggregates.Subsequently, cell suspension was centrifuged at 1000 rpm for 5 min at 4 °C.Cells were collected, and resuspended in PBS.Each sample was a pool of cells from the intestinal epithelium of 3 mice.

Single-cell RNA sequencing
Single cell capture, library preparation, sequencing and data analysis were performed by Capitalbio Technology Corporation (Beijing, China).Single cell capture and library preparation were performed following general protocols.For Seurat pipeline, cells whose gene number was less than 200, or gene number ranked in the top 1%, or mitochondrial gene ratio was more than 25% were regarded as abnormal and filtered out.Dimensionality reduction was performed using PCA, and visualization was realized by TSNE.Cells were defined based on the marker genes of each population as reported 7 .Single-cell trajectories were built with Monocle (R package) that introduced pseudotime.Genes were filtered by the following criteria: Expressed in more than 10 cells; The average expression value was greater than 0.1; Qval was less than 0.01 in different analysis.

Gut microbiota analysis by 16S rRNA gene sequencing
Total genome DNA from samples harvested from ileocecal contents was extracted using CTAB/SDS method.Following 16s rRNA V3-V4 region amplification, PCR products quantification and qualification, DNA library was constructed using the TruSeq® DNA PCR-Free Sample Preparation Kit.Sequencing libraries were generated using TruSeq® DNA PCR-Free Sample Preparation Kit (Illumina, USA) following manufacturer's recommendations and index codes were added.The library quality was assessed on the Qubit@ 2.0 Fluorometer (Thermo Scientific) and Agilent Bioanalyzer 2100 system.After the library qualified by Qubit and Q-PCR, NovaSeq6000 was used for sequencing and 250 bp paired-end reads were generated.Sequences with ≥97% similarity were assigned to the same OTUs.Shannon index was applied in analyzing complexity of species diversity, calculated with QIIME and displayed with R software.

Statistical analysis
Using Prism software for graphing and statistical analysis, the experimental results were shown as mean ± SD.Statistical significance of differences between the groups was analyzed with a t-test or one/twoway ANOVA with Šídák's multiple comparisons test.P < 0.05 denotes statistical significance.

Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Fig. 3 |
Fig. 3 | Deficiency of intestinal Lgr4 decreases lipid absorption.a-f Six-week-old male Lgr4 iKO mice and littermates were fed normal chow diet (NCD) or 60% high fat diet (HFD) for 12 weeks.Results were expressed as mean ± SD and analyzed by the t-test or one-way ANOVA.*P < 0.05 vs WT NCD.# P < 0.05 vs WT HFD.n = 3-6.a Fecal triglyceride levels.n = 4 for WT NCD, 4 for Lgr4 iKO NCD, 5 for WT HFD, and 4 for Lgr4 iKO HFD.WT NCD vs. Lgr4 iKO NCD: *P = 0.0296.WT NCD vs. WT HFD: *P = 0.0185.WT HFD vs. Lgr4 iKO HFD: # P = 0.011.b Levels of circulating triglyceride and the area under curve in response to oral administration of olive oil in NCD-fed mice.n = 4. *P = 0.0004 for AUC.c Oil red O staining of intestine 2 h after olive oil gavage and quantitative analysis.n = 4 for WT NCD and 3 for Lgr4 iKO NCD.*P = 0.0107.d mRNA levels of lipid absorption markers (Fatp4, Cd36 and Fabp2) in intestine of NCD-fed mice.n = 5 for WT NCD and 5-6 for Lgr4 iKO NCD.*P = 0.0483 for Cd36, *P = 0.0104 for Cav1.e Immunohistochemical staining of FATP4 in intestine and quantification of positive area.n = 9. *P = 0.0004(left) and 0.0075(right).f Western blot and quantification of CD36 protein levels.β-actin was used as internal control.n = 3. *P = 0.0093.g-j The mouse small intestinal epithelial cell line MODE-K cells were transfected with Lgr4 siRNA for 48 h.g Western blot and quantification of LGR4 and FATP4 protein levels.β-actin was used as loading control.n = 3. *P = 0.008 for LGR4, *P = 0.0124 for FATP4.h mRNA levels of lipid absorption markers (Fatp4, Cav1, Fabp1 and Fabp2) in MODE-K cells.β-actin was used as a reference gene.n = 3. *P = 0.0077 for Fatp4, *P = 0.0085 for Cav1.i The triglyceride level in MODE-K cells treated with mixture of oleic acid (0.6 mmol/l) and palmitic acid (0.2 mmol/l).n = 5. j Cells were treated with BODIPY-C 12 longchain fatty acid for 2 h and observed under microscope and the uptake of BODIPY-C 12 long-chain fatty acid in MODE-K cells by flow cytometry.

Fig. 6 |
Fig.6 |LGR4 regulates differentiation of ISCs via Wnt and Notch signaling pathways.a-i Six-week-old male Lgr4 iKO mice and littermates were fed normal chow diet for 12 weeks.Single cell RNA sequencing was used to obtain intestinal epithelium single cell transcriptome data from 18-week-old Lgr4 iKO mice and littermates.n = 3. *P < 0.05 vs WT. a The proportions of stem cells, TA cells, absorptive cells, goblet cells, paneth cells, enteroendocrine cells and tuft cells.b t-SNE plot showing absorptive progenitor cell marker genes expression (left) and heatmap showing UMI value (right).The transcription expression levels were calculated as UMI value.c t-SNE plot showing secretory progenitor cell marker genes expression (left) and heatmap showing UMI value of Dll1 (right).d mRNA levels of absorptive progenitor cell and secretory progenitor cell markers in intestine of NCD-fed mice.n = 4-5 for WT, and 5-6 for Lgr4 iKO .Results were expressed as mean ± SD. e Pseudotime showing Wnt target genes expression (left) and heatmap showing UMI value (right).f Western blotting detecting nuclear and cytosol levels of β-catenin protein.The relative expression level was quantified using Image J software.Results were expressed as mean ± SD. g mRNA levels of downstream genes of the Notch signaling pathway (Math1 and Hes1) detected by RT-qPCR.n = 5 for WT, and 6 for Lgr4 iKO .Results were expressed as mean ± SD. h Pseudotime showing Notch related genes expression (left) and heatmap showing UMI value (right).i Western blot and quantification of HES1 protein levels.β-actin was used as loading control.Results were expressed as mean ± SD. j Wnt and Notch related genes of the mouse small intestinal epithelial cell line MODE-K cells treated with the Notch activator VPA (P4543-25G, Sigma).*P < 0.05 vs NC. n = 3. Statistical analysis by two-way ANOVA with Šídák's multiple comparisons test.* P = 0.0001 for Axin2, * P = 0.0035 for Ccnd1, * P = 0.0003 for Hes1, * P < 0.0001 for Hey1.k mRNA levels of lipid absorption markers of MODE-K cells treated with the Wnt inhibitor IWR-1 (HY-12238, MedChemExpress).*P < 0.05 vs NC. n = 3. Statistical analysis by two-way ANOVA with Šídák's multiple comparisons test.* P = 0.0178.

Fig. 7 |
Fig. 7 | Graphic highlight of findings.Deficiency of intestinal Lgr4 reduces enterocytes selective for absorption of long-chain fatty acid, leading to reduction in lipid absorption and subsequent metabolic benefit.